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Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation of p21 Gene Expression.

Geng D, Zhang Z, Guo H - Biosensors (Basel) (2012)

Bottom Line: Here we report the development of a fish cell biosensor system for high throughput genotoxicity detection of new drugs, by stably integrating two reporter plasmids of pGL3-p21-luc (human p21 promoter linked to firefly luciferase) and pRL-CMV-luc (CMV promoter linked to Renilla luciferase) into marine flatfish flounder gill (FG) cells, referred to as p21FGLuc.Initial validation of this genotoxicity biosensor system showed that p21FGLuc cells had a wild-type p53 signaling pathway and responded positively to the challenge of both directly acting genotoxic agents (bleomycin and mitomycin C) and indirectly acting genotoxic agents (cyclophosphamide with metabolic activation), but negatively to cyclophosphamide without metabolic activation and the non-genotoxic agents ethanol and D-mannitol, thus confirming a high specificity and sensitivity, fast and stable response to genotoxic agents for this easily maintained fish cell biosensor system.A limitation for this biosensor system was that it might give false positive results in response to sodium butyrate and any other agents, which can trans-activate the p21 gene in a p53-independent manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Marine Biology, Ocean University of China, Qingdao 266003, China. doarey@126.com.

ABSTRACT
p21CIP1/WAF1 is a p53-target gene in response to cellular DNA damage. Here we report the development of a fish cell biosensor system for high throughput genotoxicity detection of new drugs, by stably integrating two reporter plasmids of pGL3-p21-luc (human p21 promoter linked to firefly luciferase) and pRL-CMV-luc (CMV promoter linked to Renilla luciferase) into marine flatfish flounder gill (FG) cells, referred to as p21FGLuc. Initial validation of this genotoxicity biosensor system showed that p21FGLuc cells had a wild-type p53 signaling pathway and responded positively to the challenge of both directly acting genotoxic agents (bleomycin and mitomycin C) and indirectly acting genotoxic agents (cyclophosphamide with metabolic activation), but negatively to cyclophosphamide without metabolic activation and the non-genotoxic agents ethanol and D-mannitol, thus confirming a high specificity and sensitivity, fast and stable response to genotoxic agents for this easily maintained fish cell biosensor system. This system was especially useful in the genotoxicity detection of Di(2-ethylhexyl) phthalate (DEHP), a rodent carcinogen, but negatively reported in most non-mammalian in vitro mutation assays, by providing a strong indication of genotoxicity for DEHP. A limitation for this biosensor system was that it might give false positive results in response to sodium butyrate and any other agents, which can trans-activate the p21 gene in a p53-independent manner.

No MeSH data available.


Related in: MedlinePlus

Validation of the endogenous p53-signaling pathway in FG cells. Examination of the responses of the transiently transformed FG cells to genotoxicant of bleomycin (30 μg/mL for 4 h) using firefly luciferase reporter plasmids of pGL3-p21-luc and pGL3-p53-luc and the Renilla luciferase internal reference plasmid of pRL-CMV. The intact FG cells (not transformed) were used as control. Data are expressed as mean ± SD.
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biosensors-02-00318-f003: Validation of the endogenous p53-signaling pathway in FG cells. Examination of the responses of the transiently transformed FG cells to genotoxicant of bleomycin (30 μg/mL for 4 h) using firefly luciferase reporter plasmids of pGL3-p21-luc and pGL3-p53-luc and the Renilla luciferase internal reference plasmid of pRL-CMV. The intact FG cells (not transformed) were used as control. Data are expressed as mean ± SD.

Mentions: As shown in Figure 3, the DNA damaging agent of bleomycin can significantly induce the over-expression of both the p53 and p21 promoter-driven firefly luciferase reporter genes in the transiently transformed FG cells. The efficient trans-activation of pGL3-p53-luc plasmid by DNA damage-induced p53 signaling pathway of FG cells indicates that the upstream regulators of the p53 protein in response to DNA damage are valid in FG cells. Similarly, the efficient trans-activation of pGL3-p21-luc plasmid showed that FG cells still held wild-type p53 proteins in spite of the fact that they had become a continuous cell line and undergone neoplastic transformation, and also p53 proteins of FG cells were able to recognize and trans-activate the promoter of human p21 gene. In contrast, extremely low background luciferase activity was detected in the untransformed FG cells. The luminescence of firefly luciferase and Renilla luciferase in the untransformed FG cells was only 66 RLU and 190 RLU, respectively, about eight to nine orders of magnitude lower than those in the transformed FG cells. It was also observed that the expression of Renilla luciferase from pRL-CMV plasmid was not DNA damage-inducible but cell number-dependent (data not shown).


Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation of p21 Gene Expression.

Geng D, Zhang Z, Guo H - Biosensors (Basel) (2012)

Validation of the endogenous p53-signaling pathway in FG cells. Examination of the responses of the transiently transformed FG cells to genotoxicant of bleomycin (30 μg/mL for 4 h) using firefly luciferase reporter plasmids of pGL3-p21-luc and pGL3-p53-luc and the Renilla luciferase internal reference plasmid of pRL-CMV. The intact FG cells (not transformed) were used as control. Data are expressed as mean ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263550&req=5

biosensors-02-00318-f003: Validation of the endogenous p53-signaling pathway in FG cells. Examination of the responses of the transiently transformed FG cells to genotoxicant of bleomycin (30 μg/mL for 4 h) using firefly luciferase reporter plasmids of pGL3-p21-luc and pGL3-p53-luc and the Renilla luciferase internal reference plasmid of pRL-CMV. The intact FG cells (not transformed) were used as control. Data are expressed as mean ± SD.
Mentions: As shown in Figure 3, the DNA damaging agent of bleomycin can significantly induce the over-expression of both the p53 and p21 promoter-driven firefly luciferase reporter genes in the transiently transformed FG cells. The efficient trans-activation of pGL3-p53-luc plasmid by DNA damage-induced p53 signaling pathway of FG cells indicates that the upstream regulators of the p53 protein in response to DNA damage are valid in FG cells. Similarly, the efficient trans-activation of pGL3-p21-luc plasmid showed that FG cells still held wild-type p53 proteins in spite of the fact that they had become a continuous cell line and undergone neoplastic transformation, and also p53 proteins of FG cells were able to recognize and trans-activate the promoter of human p21 gene. In contrast, extremely low background luciferase activity was detected in the untransformed FG cells. The luminescence of firefly luciferase and Renilla luciferase in the untransformed FG cells was only 66 RLU and 190 RLU, respectively, about eight to nine orders of magnitude lower than those in the transformed FG cells. It was also observed that the expression of Renilla luciferase from pRL-CMV plasmid was not DNA damage-inducible but cell number-dependent (data not shown).

Bottom Line: Here we report the development of a fish cell biosensor system for high throughput genotoxicity detection of new drugs, by stably integrating two reporter plasmids of pGL3-p21-luc (human p21 promoter linked to firefly luciferase) and pRL-CMV-luc (CMV promoter linked to Renilla luciferase) into marine flatfish flounder gill (FG) cells, referred to as p21FGLuc.Initial validation of this genotoxicity biosensor system showed that p21FGLuc cells had a wild-type p53 signaling pathway and responded positively to the challenge of both directly acting genotoxic agents (bleomycin and mitomycin C) and indirectly acting genotoxic agents (cyclophosphamide with metabolic activation), but negatively to cyclophosphamide without metabolic activation and the non-genotoxic agents ethanol and D-mannitol, thus confirming a high specificity and sensitivity, fast and stable response to genotoxic agents for this easily maintained fish cell biosensor system.A limitation for this biosensor system was that it might give false positive results in response to sodium butyrate and any other agents, which can trans-activate the p21 gene in a p53-independent manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Marine Biology, Ocean University of China, Qingdao 266003, China. doarey@126.com.

ABSTRACT
p21CIP1/WAF1 is a p53-target gene in response to cellular DNA damage. Here we report the development of a fish cell biosensor system for high throughput genotoxicity detection of new drugs, by stably integrating two reporter plasmids of pGL3-p21-luc (human p21 promoter linked to firefly luciferase) and pRL-CMV-luc (CMV promoter linked to Renilla luciferase) into marine flatfish flounder gill (FG) cells, referred to as p21FGLuc. Initial validation of this genotoxicity biosensor system showed that p21FGLuc cells had a wild-type p53 signaling pathway and responded positively to the challenge of both directly acting genotoxic agents (bleomycin and mitomycin C) and indirectly acting genotoxic agents (cyclophosphamide with metabolic activation), but negatively to cyclophosphamide without metabolic activation and the non-genotoxic agents ethanol and D-mannitol, thus confirming a high specificity and sensitivity, fast and stable response to genotoxic agents for this easily maintained fish cell biosensor system. This system was especially useful in the genotoxicity detection of Di(2-ethylhexyl) phthalate (DEHP), a rodent carcinogen, but negatively reported in most non-mammalian in vitro mutation assays, by providing a strong indication of genotoxicity for DEHP. A limitation for this biosensor system was that it might give false positive results in response to sodium butyrate and any other agents, which can trans-activate the p21 gene in a p53-independent manner.

No MeSH data available.


Related in: MedlinePlus